Fibroblast growth factor receptor-bearing neurons in the CNS: identification by receptor-mediated retrograde transport.

Neurons that internalize and retrogradely accumulate acidic (aFGF) or basic (bFGF) fibroblast growth factor were identified by autoradiography after injections of 125 I-aFGF or 125I-bFGF into the adult rat central nervous system (CNS). Neuronal cell bodies within the lateral hypothalamus, pedunculpontine tegmental nucleus, laterodorsal tegmental nucleus, and the paracentral dorsal tegmental nucleus accumulated 125I-aFGF. Neurons in the hippocampus, subiculum, the centrolateral, paracentral, central medial, and parafascicular thalamic nuclei, the supramammillary nucleus, and substantia nigra compacta accumulated 125I-bFGF. The pattern of neuronal labeling with 125I-bFGF in adult rats was similar to that observed in newborn guinea pigs. No 125I-FGF labeling was observed in nerve growth factor (NGF) receptor-bearing neurons, including the basal forebrain cholinergic neurons. Time-course studies indicate that 125I-FGF was internalized at the terminals and retrogradely transported to the neuronal cell bodies. Neurons were retrogradely labeled either by injection of 125I-bFGF into the lateral ventricle or by injection into innervated target tissues. Co-injection of a 250-fold excess of unlabeled FGF with the 125I-FGF abolished the neuronal labeling. Co-injection of wheat germ agglutinin (WGA), which nonspecifically blocks binding of 125I-bFGF to its receptor, also prevented neuronal labeling. These studies demonstrate that specific neuronal populations within the CNS express functional receptors for aFGF and/or bFGF; in these neurons, aFGF and/or bFGF bind specifically to these receptors, are internalized and retrogradely transported to the neuronal soma in a manner analogous to NGF. The data indicate that FGF can provide trophic support to CNS neurons by both direct and indirect mechanisms.

Receptor-mediated retrograde transport in CNS neurons after intraventricular administration of NGF and growth factors.

Radiolabel tracer techniques were used to follow the distribution of nerve growth factor (NGF) and other neuromodulatory factors after intraventricular injection. Autoradiography showed that shortly after intraventricular injection of radio-iodinated NGF (125I-NGF), substantial amounts of radioactivity had penetrated the ventricular wall surfaces; this binding was transient and nonspecific. The 125I-NGF was progressively cleared from the central nervous system (CNS), presumably via the flow of cerebrospinal fluid (CSF) into the blood. A relatively small proportion of the injected 125I-NGF was taken up by NGF receptor-positive neurons in the CNS. Retrograde accumulation of radiolabel was observed within the basal forebrain cholinergic neurons at 5 hours after intraventricular injection. Labeling intensity was maximal at 18 hours and much reduced by 30 hours. This labeling was blocked by co-injection of an excess of unlabeled NGF. Specific and saturable retrograde labeling was also observed within other NGF receptor-bearing neurons, including the prepositus hypoglossal nucleus and the raphe obscurus nucleus. When epidermal growth factor (EGF), transforming growth factor-beta 1 (TGF-beta 1), platelet-derived growth factor-AA (PDGF-AA), PDGF-BB, leukemia inhibitory factor (LIF), insulin-like growth factor-I (IGF-I), or IGF-II was radiolabeled and injected intraventricularly, specific labeling of neurons was observed for 125I-IGF-II and 125I-LIF within separate subpopulations of the dorsal and medial raphe. No retrograde accumulation within neurons was observed for EGF, TGF-beta 1, PDGF-AA, PDGF-BB, or IGF-I. This study describes an in vivo method for identifying putative neuromodulatory factors and their responsive neurons.

Serotonergic neurons in the brainstem of the wallaby, Macropus eugenii.

The organisation and cytoarchitecture of the serotonergic neurons in a diprotodont marsupial were examined by using serial sections of the brainstem processed for serotonin immunohistochemistry and routine histology. The topographic distribution of serotonergic neurons in the brainstem of the adult wallaby (Macropus eugenii) was similar to that of eutherian mammals. Serotonergic neurons were divided into rostral and caudal groups, separated by an oblique boundary through the pontomedullary junction. Approximately 52% of the serotonergic neurons in the wallaby brainstem were located in the rostral midline nuclei (caudal linear nucleus, dorsal, median, and pontine raphe nuclei and the interpeduncular nucleus), whereas 21% were found in the caudal midline region (nuclei raphe magnus, obscurus, and pallidus). The remaining serotonergic neurons (27%) were located in more lateral regions such as the pedunculopontine tegmental nuclei, the supralemniscal nuclei (B9 group), and the ventrolateral medulla. The largest serotonergic group, the dorsal raphe, contained one-third of the brainstem serotonergic neurons and showed five subdivisions, similar to that described in other species. In contrast, the median raphe did not show clear subdivisions. The internal complexity of the raphe nuclei and the degree of lateralisation of serotonergic neurons suggest that the wallaby serotonergic system is similar in organisation to that described for the cat and rabbit. This study supports the suggestion that the serotonergic system is evolutionally well conserved and provides baseline data for a quantitative study of serotonergic innervation of the developing cortex in the wallaby.

Fibrin network structure: modification by platelets.

Platelets play a fundamental role in haemostasis and thrombosis. They are known to undergo characteristic changes including release of subcellular material during clotting. The effect of subcellular platelet material on fibrin network structure, however, has not previously been investigated. Using opacity ratio, syneresis, permeation and electron microscopy it was found that subcellular platelet material extracted into NaCl is able to influence fibrin network structure of clots made from purified fibrinogen as well as platelet-poor plasma. Such clots had higher opacity ratio, reduced syneresis and lower permeability than control clots. Further, the responsible platelet material is heat labile and is released from the platelets during their aggregation with several common aggregating agents. Morphometric analysis of transmission electron micrographs has shown that fibrin fibres in plasma clots made in the presence of platelet subcellular material are thinner than those in control clots. In addition, plasma clots made in the presence of platelet extract had a higher resistance to fibrinolytic digestion than control clots. Thus, platelets play a hitherto undescribed role in regulating fibrin network structure.

The role of coronary artery disease in complications of abdominal aortic aneurysm surgery.

Coronary artery disease (CAD) is a major cause of morbidity and mortality after elective surgical repair of abdominal aortic aneurysm (AAA). The aim of this study was to determine the relationship between the extent of CAD observed in coronary angiograms (more than 50% stenosis) and the frequency of postoperative myocardial ischemic complications in a consecutive series of 84 patients who underwent elective AAA repair. Ninety-four percent of the patients with clinical evidence of CAD had significant disease as observed in coronary angiograms and eight patients had left main CAD. Seventy-two patients underwent AAA repair with a mortality rate of 1.4%; five patients had preliminary myocardial revascularization, and AAA surgery was not recommended for four patients because of severe cardiac disease. Postoperative myocardial ischemic complications occurred in 13.4% of the patients who had undergone surgery--almost exclusively in patients with clinical evidence of CAD. Both myocardial ischemia and preoperative intervention were more frequent in patients with double- or triple-vessel disease than in patients with less extensive disease. Patients with symptoms and with double- or triple-vessel CAD have a high risk of developing myocardial ischemia after AAA surgery. Preliminary myocardial revascularization may be beneficial in this group of patients.

Comparison of wheat germ agglutinin-horseradish peroxidase and biotinylated dextran for anterograde tracing of corticospinal tract following spinal cord injury.

Established methods for monitoring regeneration of the corticospinal tract involve anterograde labelling of the cortical motor neuron. While wheat germ agglutinin-horseradish peroxidase conjugate has been used to anterogradely label these neurons, we demonstrate that this technique may not completely label the whole axon and fine terminal processes when this tracer is administered in dried form. An alternative method is described for anterograde labelling of cortical motor neurons using biotinylated dextran. This tracer may be applied by either microinjection of 10% biotinylated dextran or implanting small globules of the dried tracer into the motor cortex. While more laborious, microinjection results in better anterograde labelling than implantation of dried biotinylated dextran. A procedure is also described for preparing serial coronal sections through the entire spinal cord and thaw-mounted on a minimum number of slides. The labelled nerve processes in these tissue sections can be visualised in the spinal cord under a fluorescent microscope following incubation with cy3-streptavidin complex. Permanent labelling of the biotinylated nerve processes is achieved by incubation of tissue sections with streptavidin-horseradish peroxidase conjugate followed by stringent washes and staining with tetramethylbenzidine. Use of tetramethylbenzidine allows resolution of a greater number of finer labelled processes than diaminobenzindine and allows clear visualisation of individual regenerating corticospinal tract processes. Using these procedures, we demonstrate that the corticospinal tract is completely lesioned by a standardised contusion spinal cord injury produced by the New York University weight-drop device.

Preoperative blood tests in prediction of postoperative deep vein thrombosis.

Fifty six patients undergoing elective abdominal surgery were investigated preoperatively with tests of coagulation, platelet function and fibrinolysis. Ten patients developed postoperative deep vein thrombosis, detected by the labelled fibrinogen uptake test and confirmed by ascending phlebography. None of the tests showed a statistically significant difference between the group mean of patients who developed DVT and of those who did not. Potential discriminators were used to derive a prognostic index for prediction of patients who would develop postoperative DVT. An index based on two preoperative blood tests i.e. three hour fibrin digestion and APTT had a successful prediction rate of 59 percent.

Peritoneal surface-active material in continuous ambulatory peritoneal dialysis (CAPD) patients.

Phospholipids have been demonstrated to be present in the peritoneal dialysis effluent of 34 patients on continuous ambulatory peritoneal dialysis (CAPD). The phospholipids present have been characterized by chromatography and their relative concentrations are fairly consistent from patient to patient. The predominant phospholipid is phosphatidylcholine (81%). Surface activity of this phospholipid has been demonstrated. The concentration of total phospholipid correlates with the time the patient had been on CAPD. It is lower in those patients who have been on dialysis longer.

Phagocytosis of bacteria by olfactory ensheathing cells and Schwann cells.

Opportunistic bacterial infections of the nasal cavity could potentially lead to infection of the brain if the olfactory or trigeminal nerves are colonised. The olfactory nerve may be a more susceptible route because primary olfactory neurons are in direct contact with the external environment. Peripheral glia are known to be able to phagocytose some species of bacteria and may therefore provide a defence mechanism against bacterial infection. As the nasal cavity is frequently exposed to bacterial infections, we hypothesised that the olfactory and trigeminal nerves within the nasal cavity could be subjected to bacterial colonisation and that the olfactory ensheathing cells and Schwann cells may be involved in responding to the bacterial invasion. We have examined the ability of mouse OECs and Schwann cells from the trigeminal nerve and dorsal root ganglia to phagocytose Escherichia coli and Burkholderia thailandensis in vitro. We found that all three sources of glia were equally able to phagocytose E. coli with 75-85% of glia having phagocytosed bacteria within 24h. We also show that human OECs phagocytosed E. coli. In contrast, the mouse OECs and Schwann cells had little capacity to phagocytose B. thailandensis. Thus subtypes of peripheral glia have similar capacities for phagocytosis of bacteria but show selective capacity for the two different species of bacteria that were examined. These results have implications for the understanding of the mechanisms of bacterial infections as well as for the use of glia for neural repair therapies.

Endogenous chicken nerve growth factor from sheath cells is transported in regenerating nerve.

We report the presence of endogenous nerve growth factor (NGF) in chicken peripheral nerve. The molecule has been detected with antibodies to mouse salivary gland NGF, using immunohistochemical and immunoelectrophoretic techniques. Previous studies have shown that these antibodies inhibit the survival activity of extracts of chicken peripheral nerve. The NGF accumulated distal, but not proximal, to a ligature placed on a peripheral sympathetic nerve demonstrating that it was retrogradely transported. This transport was detected in intact nerve fibers as well as in nerves from which the peripheral target had been ablated 6 hr or 7 days previously. The results indicate that avian NGF is present in adult chicken peripheral nerves and that this molecule shares antigenic determinants with the mouse molecule. The results further demonstrate that regenerating neurons retrogradely transport NGF supplied by cells within the peripheral nerve (presumably Schwann). The possibility that these cells also provide NGF to intact neurons is discussed.

Stimulation of corticospinal tract regeneration in the chronically injured spinal cord.

Acute spinal cord injury models have proved popular in studies aimed at identifying factors capable of influencing axonal regeneration within the central nervous system. In these models, the test factors (e.g. graft tissues or cells, antibodies, growth factors, etc.) are typically administered at the time of spinal cord injury. In this study, we use a rat chronic spinal cord injury model to identify possible factors which can stimulate regeneration of the chronically lesioned corticospinal tract axons. We demonstrate that surgical grafting of segments of autologous, preligated sural nerve, into the syrinx, stimulates sprouting and regeneration of the corticospinal tract as evidenced by the presence of anterograde labelled corticospinal tract processes within the cavity walls two or more weeks after treatment. Regrowing corticospinal processes were not observed within control animals. The anterogradely labelled corticospinal tract axons were found exclusively within the central grey tissue comprising the cavity walls with no regrowing corticospinal process observed within the white matter. A similar pattern of regeneration was observed following injection into the cavity of a suspension of minced autologous preligated sural nerve. Evidence of corticospinal tract regeneration was seen when either wheat germ agglutinin--horseradish peroxidase or biotinylated--dextran was used as an anterograde tracer. These data demonstrate that the chronically injured cortical motor neurons retain the capacity to regenerate for extended periods and that regeneration can be stimulated using grafts of minced, preligated autologous peripheral nerve tissue.

Basic fibroblast growth factor: receptor-mediated internalization, metabolism, and anterograde axonal transport in retinal ganglion cells.

Basic fibroblast growth factor (bFGF) was radiolabeled and used in axonal transport studies to determine whether certain neuronal populations express functional receptors for bFGF. Unlike 125I-NGF, 125I-bFGF was not retrogradely transported in the adult rat sciatic nerve or from iris to trigeminal ganglion or superior cervical ganglion. However, after intraocular injection of 125I-bFGF into the posterior chamber of the eye of adult rats, radioactivity was detected within the retinal ganglion cell projections. This radioactivity was localized to the ipsilateral optic nerve and in the contralateral lateral geniculate body and the contralateral superior colliculus by using autoradiographic techniques. Direct measurement of the radioactivity in dissected brain regions was used to study the process of 125I-bFGF uptake and transport by retinal ganglion cells. The uptake and transport were specific for biologically active bFGF since neither denatured, biologically inactive 125I-bFGF nor 125I-NGF was taken up and transported. The uptake and transport of 125I-bFGF were saturable phenomena since they were blocked in the presence of excess, unlabeled bFGF. Wheat germ agglutinin, but not heparinase, blocked uptake and transport of 125I-bFGF, a finding that is consistent with the uptake being mediated by high-affinity bFGF receptors. Radioactivity from 125I-bFGF was transported in retinal ganglion cell axons in an anterograde direction at a maximum rate in excess of 1.7 mm/hr. No specific retrograde transport of bFGF to the retina was detected after 125I-bFGF was injected into the superior colliculus. The radioactivity from 125I-bFGF that accumulated in the superior colliculus was lost from this tissue with a half-life of about 22 hr. Autoradiography of proteins separated by SDS-PAGE demonstrated that 125I-bFGF was not substantially degraded in the retina after internalization within retinal ganglion cells. During anterograde transport, however, 125I-bFGF underwent limited proteolytic cleavage resulting in 3 prominent 125I-bFGF derivatives of molecular weights greater than 7000 Da. Although these were the major radioactive species recovered from the superior colliculus after intraocular injection, some intact 125I-bFGF was also detected within the innervated target. These results indicate that retinal ganglion cells express high-affinity receptors for bFGF, that these receptors mediate the internalization of bFGF, that internalized bFGF undergoes limited proteolytic cleavage, and that bFGF and its derivatives are anterogradely transported to the lateral geniculate body and the superior colliculus. These data raise the possibility that bFGF or its derivatives may act as an anterograde trophic factor in the visual system, a system that is known to undergo anterograde transneuronal cell death.

Chicken NGF and non-NGF trophic factor synthesis and release by sciatic nerves in vitro.

The time course of production and release of nerve growth factor (NGF) and non-NGF neuronotrophic factors for sympathetic neurons by chicken and rat sciatic nerves in culture was examined. These tissues actively synthesize and release neuronotrophic activity as metabolically poisoning nerves with azide dramatically reduced the amount of trophic activity released into the culture medium. The sustained release of this activity also was shown to be dependent on the presence of low-molecular-weight dialysable molecules present in foetal calf serum and amniotic fluid from day 11 chicken embryos. Affinity-purified antimouse NGF antibodies were used to show that sciatic nerves in culture release both NGF and non-NGF trophic factors. These antibodies inhibited all bioactivity of both mouse NGF and of a partially purified preparation of chicken NGF. Immunoblot studies confirm that the antibodies recognize both rodent and avian NGF. Excess antibody inhibited only about 50% of the trophic activity in media conditioned over rat or chicken nerves for the first 24 hr. Relatively similar amounts of this non-NGF trophic activity were released throughout 6 days in culture, and this trophic activity kept sympathetic neurons alive in culture in the absence of NGF for more than 4 days. NGF levels were quantified with a two-site enzyme-linked immunoassay and found to parallel changes in NGF bioactivity. Rat nerves released increasing amounts of NGF with time in culture. Whole chicken sciatic nerves, however, released decreasing amounts of NGF with time in culture, but when these nerves were desheathed by removal of the epineurium and attached tissue, the pattern of NGF release was similar to that observed in the smaller rat sciatic nerves. These studies therefore characterize antibodies recognizing chicken NGF, demonstrate that peripheral nerve tissue synthesize trophic factors other than NGF, and identify factors that influence NGF synthesis.

Nerve growth factor immunohistochemistry and biological activity in the rat iris.

Nerve growth factor (NGF) production in the cultured rat iris was examined using immunohistochemistry and bioassay of irides and conditioned media. NGF immunoreactivity increased steadily with days in culture so that the intensity of staining was maximal after 6 days of culture. The localization was shown to be sensitive to the presence of cross-linking fixatives such as formaldehyde and glutaraldehyde and this effect was only partially alleviated by the use of very high concentrations of antibodies. NGF immunoreactivity was localized in Schwann cells and possibly nerve axons, but with no antigen detectable in smooth muscle fibres. Media conditioned over irides initially supported a high percentage of dissociated sympathetic neurons, but the number supported decreased with time in culture until day 4. Moreover, the use of antibodies to NGF allowed the detection of at least two types of neuronotropic activity, NGF accounting for at least 94% of the total trophic activity present after 4 days of culture. These findings provide support for the proposal that Schwann cells produce NGF and question the accepted hypothesis that the molecule is produced by smooth muscle fibres as a peripheral maintenance factor for sympathetic and sensory nerves. The results also suggest that two survival factors may be involved in the regulation of sympathetic function.

Arterial reconstruction extending below the popliteal bifurcation.

Results are reported for 172 anastomoses below the popliteal artery in patients almost exclusively suffering from severe distal ischaemia with an in-hospital mortality of 7.6 per cent. The patency rate in survivors was 83 per cent on discharge and 39 per cent at 3 years. There were 37 major amputations following 172 operations and 15 following 47 operations on diabetics. As most of the patients would have lost their limbs if arterial repair was not undertaken this constitutes a considerable salvage rate. Diabetes mellitus does not appear to influence the results infavourably, so patients suffering from this disease should be considered for operation.

Immunohistochemical evidence for the distribution of nerve growth factor in the embryonic mouse.

The distribution of nerve growth factor-like immunoreactivity has been examined in the embryonic mouse with special reference to the CNS. The intensity of the immunoreactive stain was found to be greatest on embryonic days 15 and 16. The antigen is widespread and present in high concentrations in both the PNS and CNS. Most intense staining was detected in cranial nerve tracts, hippocampus, developing white matter of the spinal cord and tegmentum. Lower intensities were found within diencephalic regions, spinal cord grey matter, medullary fibre tracts and cerebellum. These results support the increasing evidence suggesting that the trophic molecule nerve growth factor has an important role to play in the development of central as well as peripheral neurons.

The use of regional, low dose streptokinase in recently thrombosed arterial grafts.

Between August 1983 and January 1985, 20 patients aged 33-77 years, with occluded lower limb bypass grafts, were on 23 occasions treated with streptokinase via intra-arterial infusion. Streptokinase (5000 units/h) was effective in clearing occluded grafts in 15 patients on 16 occasions. The median duration of occlusion in these patients was 5 days and the median duration of streptokinase infusions was 24 h. Completion angiography following streptokinase thrombolysis revealed five graft stenoses and 12 outflow stenoses or occlusions. In two grafts no cause for graft failure could be identified. These results permitted the surgeon to make an accurate pre-operative assessment of the definitive therapy required to ensure graft patency.